Current immunoassays such as enzyme linked immunosorbant assay (ELISA) performed in 96-well microtiter plate requires a minimum sample volume of 50 μL per well, i.e. 150 μL to perform the test in triplicate. In addition, the sample can only be tested for a single chemical or biochemical (e.g. protein, toxin, drug) in a given well. Commercial alternatives for the multiplexed detection of several biochemicals exist (e.g. Luminex (Life Technologies Corp.)) but are expensive, require extensive preparative steps and relatively large sample volumes. There is a need for more sensitive, miniaturised and faster multiplexed assays. In addition, the immunoassay format limits the spatial resolution over the enzymatic reaction, i.e. the entire well turns “positive”. This makes difficult to address multiples analytes in a single sample. Sample volumes become problematic. Colorimetric microarray-in-wells have been developed but lack sensitivity, and require expensive instrumentation (e.g. optics, laser scanner, etc.).
Electrochemical sensor platforms have been previously reported [see e.g., Wan, Y., et al., Development of electrochemical immunosensors towards point of care diagnostics. Biosensors and Bioelectronics, 2013. 47(0): p. 1-11. Ley, C., et al., An electrochemical microtiter plate for parallel spectroelectrochemical measurements. Electrochimica Acta, 2013. 89(0): p. 98-105. Piermarini, S., et al., Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection. Biosensors and Bioelectronics, 2007. 22(7): p. 1434-1440. Tang, T.-C., A. Deng, and H.-J. Huang, Immunoassay with a Microtiter Plate Incorporated Multichannel Electrochemical Detection System. Analytical Chemistry, 2002. 74(11): p. 2617-2621. Castillo, J., et al., Glutamate detection from nerve cells using a planar electrodes array integrated in a microtiter plate. Biosensors and Bioelectronics, 2005. 20(10): p. 2116-2119]. However, the existing electrochemical sensor platforms are singleplex, i.e. only one electrode per well, dedicated to the measurement of only one biochemical. The disposable electrode arrays are comparatively costly, as high-end electrodes arrays are ideally photolithographically microfabricated under clean room environment. In addition, diffusion of the oxidized substrate to neighboring electrodes can cause severe background current and lead to a number of errors in the interpretation of the results.
The sensitive detection of pathogens/pathogen fragments/endotoxins with a sensitivity equal to 1 CFU/mL (CFU=colony forming unit) is a challenging task. Most systems rely on large equipment and tedious analytical procedures. The ability to detect pathogens at those levels would enable the development of a companion diagnostic able to provide a rapid answer on the level of contamination present in a sample. Accordingly, there is a need to develop a method or approach that is sensitive enough to detect low concentrations of analyte and is also versatile enough to detect multiple analytes simultaneously.